The photoreceptor is the most metabolically active neuronal cell in the human body; oxygen consumption at the inner segment of the photoreceptors increases upon dark adaptation, mainly because of the increased ATP requirements needed to maintain the dark current. Since the oxygen consumption at the inner segment of the photoreceptor increases approximately 1.5-3 times upon dark adaptation, we expect a proportionate increase in CO2 generation and the subsequent increase in CO2 at the subretinal space. The accumulation of CO2 within the subretinal space (SRS) causes acidosis which is detrimental to the health of surrounding cells (i.e., Muller cells, photoreceptors, and RPE), thus metabolic CO2 must be quickly dissipated from the SRS. We hypothesize that a large fraction of this CO2 load is dissipated by diffusion to the choroidal blood supply, and that this process is mediated by the RPE. In this study, we describe the transport of CO2 across the RPE, which involves multiple ion-transport mechanisms that consequently increase fluid-absorption across the RPE. [unreadable] [unreadable] This project entails the study of the ion-transport proteins in the hfRPE that are involved in light-dark transition in the eye. First, we show that CO2 flux across the apical membrane is higher compared to CO2 flux across the basolateral membrane. We investigated the possibility that CO2-flux across the apical membrane is mediated by aquaporin 1, which has high mRNA expression levels in the hfRPE cultures and is found at apical membrane of the rat RPE. However, pH-imaging experiments showed that this was not the case in the hfRPE. We investigated to see if the difference in apical and basolateral membrane CO2 flux is caused by the difference in total exposed surface area in the RPE (the apical membrane has approximately 3-10 times larger surface area than the basolateral membrane). An experiment that involves weakening the tight junction to allow CO2 to extend further to the apical membrane supported this hypothesis. [unreadable] [unreadable] AE2 activity is reduced when the apical bath is perfused with Ringers equilibrated with 13% CO2 because AE2 is known to be inhibited under acidic conditions. Since, a CO2 load at the apical membrane necessitates an increased HCO3 transport across the basal membrane. We present pH-imaging and electrophysiology data to confirm the presence of an electrogenic Na+/HCO3- co-transporter (NBC) at the basal membrane of the RPE: (1) reducing HCO3o at the basal bath caused a TEP-rise that corresponds to the depolarization of the basal membrane, (2) the TEP-rise was DIDS-sensitive, (3) the TEP-rise was absent in zero-Na conditions. Therefore, this basal Na/HCO3 co-transporter may mediate HCO3 efflux at the basolateral membrane. With electrophysiology experiments, we also show that increasing apical CO2 from 5% to 13% increases basal NBC activity, while decreasing CO2 load at the apical membrane (from 5% to 1%) inhibited basal NBC activity. Since the conversion of CO2 to HCO3 is catalyzed by carbonic anhydrase II, we did an experiment to show that basolateral membrane Na/HCO3 co-transporter activity is inhibited by a potent carbonic anhydrase inhibitor (dorzolamide). [unreadable] [unreadable] Although the basal Na/HCO3 co-transporter activity was dependent on CO2 load at the apical membrane, how much of HCO3 is supplied by CO2-HCO3 conversion? With TEP-recordings, we show that basolateral co-transporter activity was partially inhibited when the apical pNBC1 was blocked with DIDS. This suggests that the apical Na/HCO3 co-transporter (pNBC1) provides part of the HCO3-supply necessary for basal Na/HCO3 co-transporter activity. We also showed that the main substrate for the basolateral Na/HCO3 co-transporter is HCO3; inhibiting apical Na-transport pathways such as the Na/K ATPase, Na/K/2Cl co-transporter, and Na/H exchanger did not affect basolateral Na/HCO3 co-transport activity. [unreadable] [unreadable] We showed that CO2 affects multiple ion-transporters that ultimately increases net Na, Cl, and HCO3 absorption across the RPE. Since fluid flows with an osmotic gradient, the increase in solute transport would enhance the steady-state fluid absorption across the RPE. The CO2-induced increase in fluid-absorption may have an important physiological role because the rate of metabolic water production at the retina (as calculated based on the geometry and oxygen consumption rate of the retina) is approximately 10% of the steady state fluid absorption across the human RPE. Therefore failure to remove water from the subretinal space can potentially cause retinal detachment.